1. Field of the Invention
The present invention relates to fluid analysis, and more particularly to apparatus for body fluid analysis using optical materials.
2. Background
One method of analyzing a fluid for specific properties such as the presence and concentration of specific moieties is by analyzing changes in light directed through a surface-borne reagent to which a sample of the fluid is brought into contact. This is achieved by utilizing a reagent which produces an optically detectable physical or chemical change upon contact with one or more targeted analytes in the fluid. This method of analyzing fluids is especially effective when a colorimetric reaction is effected in the sample or on a surface in contact with the sample, wherein a color change relates to one or more specific analytes in the fluid. Such a means includes generally a specific source of light of a desired frequency tuned to the spectral range of the colorimetric reaction, and also a detector for such a light. One approach in common use is to impregnate a microporous sheet-like member with a color-producing reagent. Typically, the microporous sheet-like member is a membrane whose composition comprises a “nylon” or polyamide polymer. A fluid sample, such as of blood, is deposited on one surface of the membrane, resulting in a color change reaction with the resident reagent, and the color shade and intensity are read by means of a light beam reflected from the opposite face of the membrane. Another, increasingly contemplated, method of conducting these types of analyses is by use of optic fibers as waveguides for a light beam, both to introduce a light beam from a light source into the fluid sample and as a conduit for returning light from the fluid sample to a detector. Changes in the light beam's spectral pattern, or more often in the intensity of the beam at a specific frequency, can be correlated in some manner to the concentration of a specific analyte present in the fluid sample. Application of this concept in the field of glucose measurement in body fluids is found, for example, in U.S. Pat. No. 6,157,442. This approach customarily uses an analyte-reactive reagent or combination of reagents that undergoes a color change or develops one or more colored reaction products. Selective absorption of light at one or more frequencies associated with the spectral range of the colored reaction products occurs, resulting in measurable, (i.e., quantifiable) alterations in the light beam being accessed and evaluated by a suitable detection system. In some cases, such analyses may be based on light frequencies that occur outside the normal human range of visible light, thus entailing other than “colored” reaction products. The analyte-reactive reagent is advantageously bound or otherwise located on a surface of an optic fiber, in effect making the optic fiber into a sensor probe. For instance, an analyte-reactive reagent can be positioned on the distal tip of an optical fiber waveguide, the other end of the optic fiber being attached to an apparatus or assembly that includes a suitable detector. A suitable light source may be located extraneous to the optic fiber sensor, or brought to the fluid sample by means of the optic fiber sensor itself, or brought to the fluid sample through another optic fiber waveguide bundled with the optic fiber sensor.
Some difficulties are inherent in the use of optic fiber sensors for fluid analyses. For instance, the distal tip of an optic fiber probe presents very little surface area for attachment of an analyte-reactive reagent, resulting in a very minimal effect when assayed by means of changes in a light beam shone therethrough. Accuracy and reliability of analyses can be severely compromised by the lack of enough reagent to effect significant changes in a sensor's light beam. Another difficulty resides in designing a suitable arrangement whereby a light beam can be directed through a colored reaction product, captured by an optic fiber probe, and directed to a light detector in a reliable manner. An optimal approach would seem to be the operation of a single optic fiber probe as both the conduit for a light beam from a source into the fluid sample and the conduit for light returning from the fluid sample into the optic fiber and from thence to a suitable detection system. However, the proportion of returned light from the fluid medium adjacent the distal tip of a fiber optic waveguide is normally very poor, almost to the point of being nonexistent. When the tip is textured such as disclosed in U.S. Pat. No. 5,859,937, surface area for attachment of analyte-reactive reagents is greatly increased; also, light return is mildly enhanced by the texturing, such that a few percent of the light is recaptured by the optical fiber. Nevertheless, most of the light is lost by emanation from the surface area of the tip of even a textured tip optic fiber waveguide. The option of directing a beam of light down an optic fiber probe to a distal tip having a high surface area for disposition of a diagnostic reagent is still compromised by the loss of light signal and the minimal return of light from the distal tip through the same optic fiber to a detector.
In this regard, returned light may occur in two ways, by reflection and by reflectance. Reflected light is that portion of the light beam that, in traveling to the tip, encounters an interface between the polymer matrix and the environment (fluid sample, surface-deposited diagnostic chemistry, etc.), and which bounces back from the interface rather than crosses it. Cladding along the periphery of most optic fiber strands is designed to cause reflection of internal light beams, but no such cladding is present on a distal tip of a fiber optic probe, and a very high proportion of any light being beamed down the length of the optic fiber strand will customarily escape the confines of the optic fiber strand at that tip. Reflectance, however, refers to that portion of light that enters into a sample medium such as powder or paper, and re-emerges in the general direction of the source, where the spectral nature of the light has likely been altered by contact with the sample medium. In the present instance, the zone of analyte-reactive reagent in contact with the fluid sample represents the sample medium rather than paper or powder. The phenomenon of reflectance is treated at some length in the publication titled “Reflectance Spectroscopy: Applications of the Kubelka-Munk Theory to the Rates of Photoprocesses of Powders” by E. L. Simmons, in Applied Optics, Vol. 15, No. 4, April 1976, pages 951-954, in which also reference is made to the original publication by Kubelka and Munk in a German periodical in 1931. Such light will commonly be changed by absorption of some frequencies to varying degrees by the medium. Some patent publications loosely refer to reflectance as “reflected” light, and vice versa some patent publications refer to reflected light as “reflectance”.
In the analysis of analytes by a reagent combination disposed on the tip of an optic fiber probe, the light of interest for analytical determinations is mostly lost from the tip of the optic fiber, thus requiring some means of collection of such light by apparatus external to the fiber tip. Reflectance light re-transmitted through the optic fiber from the tip is normally of such weak intensity as to greatly lower the sensitivity of the optic fiber probe in potential analytical applications. Collecting the light by means external to the optic fiber tip may overcome this difficulty but necessarily results in more complexity of design and higher cost for analytical applications, as well as introducing possible artifacts. The need exists for improved sensors wherein reflectance can be captured effectively by the fiber optic probe itself.
Similar difficulties are encountered even with sensors employing sheet-like members such as polyamides alluded to earlier in this section. When such sensors are wetted by aqueous fluid samples to be analyzed, the wetting in itself tends to make the microporous member significantly more transparent optically, and needing enhanced reflectance for optimum performance.
An additional problem encountered with these sheet-like members is the fact that the microporous polyamide membranes used for sensor construction, particularly positively charged nylon membranes (as disclosed in U.S. Pat. No. 6,420,128, which is incorporated herein in its entirety by reference thereto), are themselves hemolytic toward erythrocytes in samples of whole blood. This releases hemoglobin, which migrates far more freely through the sample matrix than erythrocytes, and generates interferences in the subsequent colorimetric analyses. In the case of some color-producing reagents—and particularly those based on tetrazolium salts—the hemoglobin can actually react with the dye system to produce color bodies, just as would blood glucose. Since the primary use of such sensors is the determination of glucose concentrations in whole blood of diabetic patients, this drawback is of major concern. One approach has been to add a porous transport layer atop the membrane to filter out erythrocytes, such as the “double-layer” embodiment disclosed in U.S. Pat. No. 5,789,255, which is incorporated herein in its entirety by reference thereto. Another approach has been to modify the color change reagent with a soluble acrylic acid polymer, also disclosed in this same reference. Nitrite salts have also been employed in tetrazolium-salt-based diagnostic strips, wherein the nitrite binds to the released hemoglobin, thus suppressing the interfering dye formation caused non-enzymatically by the hemoglobin, as disclosed in U.S. Pat. No. 6,200,773, which is incorporated herein in its entirety by reference thereto. These difficulties and subsequent approaches illustrate an ongoing need for optical sensors wherein not only enhanced reflectance is valuable, but also wherein hemolysis of erythrocytes in body fluid samples does not occur or can be reduced to such a low background level as not to develop any significant interference in the enzymatic analysis of, say, blood glucose.